ABSTRACT: Over two-thirds of children and nearly all adults worldwide are affected by oral biofilm diseases, such as dental caries (i.e. tooth decay), resulting in billions of dollars of healthcare costs annually. The main microbial pathogen associated with this disease, Streptococcus mutans (S. mutans), forms biofilms (i.e. dental plaque) containing an exopolysaccharide (EPS) matrix and acidic microenvironments capable of dissolving enamel at the tooth surface. Current treatments exhibit a technological gap because they are poorly retained within EPS due to salivary clearance. To overcome this gap, the long-term goal of this research is to develop anti-biofilm drug delivery approaches. Specifically, these approaches will be capable of enhanced drug retention within oral biofilms and pH-responsive drug release within acidic microenvironments where enamel dissolution and cavity development occur. Prior collaborative work between the Sponsor?s and Co-sponsor?s labs has created polymer nanoparticle carriers (NPCs) that enhance the anti-biofilm effects of farnesol in vitro and in vivo. However, the current approach has limitations: 1) the pKa value of the existing NPC (? 6) is poorly aligned with biofilm pH values (? 5.5), so drug release begins at pH ~6, which may lead to premature release due to common oral pH variability; 2) the NPC exhibits modest control over pH-responsive drug release kinetics in pathogenic, acidic microenvironments (pH ? 5.5) versus normal oral pH (~7.2); and 3) NPC anti-biofilm efficacy has only been shown using farnesol although multiple drugs, including myricetin and apigenin, synergistically disrupt biofilms when combined with farnesol. The central hypothesis of this proposal is that modifying the NPC polymer composition and establishing the NPC capability to simultaneously load multiple synergistic drugs will improve anti-biofilm effectiveness in vitro and in vivo. Two aims will be developed. Aim 1 is to modify NPC polymer composition to control the pH at which drug release occurs to improve biofilm disruption in vitro and in vivo. For Aim 1, we will modify the NPC polymer composition using different monomers with pKa values more closely aligned with oral biofilm pH values (pH ?5.5) and incorporate an acid-degradable crosslinker to create a physical barrier for drug release at neutral pH. Anti-biofilm effectiveness of NPCs with the greatest pH-responsive characteristics will be tested using established in vitro and in vivo oral biofilm models. Aim 2 is to establish NPC capability to load and release multiple synergistic anti-biofilm drugs and that co-loading NPCs improves biofilm disruption in vitro and in vivo. For Aim 2, we will investigate molecular interactions indicative of loading and release between known anti- biofilm drugs (e.g. myricetin, apigenin, and farnesol) and the existing NPC to characterize which drug classes load and release successfully. The anti-biofilm efficacy of successfully co-loaded drugs will be investigated using in vitro and in vivo models. This work is significant because it will move this approach closer to a promising, clinically relevant therapeutic platform to prevent dental caries and other oral diseases.